Tracking a misclassified pathogen: genomic and epidemiological features of Vibrio paracholerae

The genus Vibrio encompasses globally relevant pathogens, of which Vibrio cholerae is the best known due to its role in cholera. Closely related species within the Cholerae clade - Vibrio paracholerae, Vibrio metoecus and Vibrio tarriae - were long misclassified as non-O1/O139 Vibrio cholerae. The objective of this study was to analyse all 13,000+ available V. cholerae genomes in GenBank to determine the presence of species from the Cholerae clade. Genome-wide analyses using Mash, whole-genome-based Average Nucleotide Identity and digital DNA-DNA hybridization reclassified 190 unique genomes as V. paracholerae, while V. metoecus and V. tarriae were not detected. Phylogenomic analyses revealed that V. paracholerae forms distinct lineages, spanning clinical, environmental and animal sources over a period of more than a century. Virulence profiling revealed the absence of cholera toxin and toxin-coregulated pilus; however, most genomes exhibited other virulence factors, including haemolysins, RTX toxins, cholix toxin and a conserved type VI secretion system. Resistome analysis revealed multiple antibiotic resistance genes, several of which were embedded within superintegron regions, reinforcing the role of V. paracholerae as a reservoir of resistance determinants. Importantly, we identified five putative gene markers with high sensitivity and specificity for discriminating the two species, providing a tool for diagnostic applications and epidemiological surveillance. These findings reveal an unsuspected epidemiological scenario for V. paracholerae, which should be considered in clinical monitoring and public health strategies involving the Cholerae clade.